Epstein Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a number of cancers including Burkitt lymphomas and lymphomas in transplant recipients. We have been studying EBV DNA in the blood of transplant recipients or other immunocompromised persons, patients with chronic active EBV disease, and patients with diseases in which EBV may be a cofactor. EBV infects B lymphocytes and establishes a latent infection in these cells in healthy persons. We developed a new technique in which we combined antibody staining to identify individual cell types in the blood and in situ hybridization to measure both (a) the cells types in the blood infected with EBV, and (b) the number of copies of EBV in infected cells from patients with high levels of EBV DNA in the blood. In patients with infectious mononucleosis we found that a large number of B cells were infected, each with a small number of copies of EBV. In other patients with high levels of EBV in the blood, we found that in addition to EBV in B cells, we detected EBV in plasmablast/plasma cells in the blood of 50% of patients, in monocytes or T cells in a small proportion of patients, and in "non-B, non-T, non-monocytes" in 70% of patients. The mean number of EBV copies in B cells was significantly higher than in plasmablast/plasma cells. Although we detected CD21, the EBV B-cell receptor, on EBV-infected B cells, we could not detect it on virus-infected T cells. This implies that a different receptor allows the virus to enter T cells. These findings expand the range of cell types infected in the blood. Determining the number of EBV genomes per cell and the type of cells infected in patients with high EBV loads may provide additional prognostic information for the development of EBV lymphoproliferative diseases. The new technique we developed may be useful for studies of other virus infections in the blood. Chronic active EBV disease (CAEBV) is an often fatal disease in which patients have markedly elevated levels of antibody to EBV or EBV DNA in the blood, and infiltration of tissues with EBV-infected lymphoocytes. We reviewed our experience with CAEBV during the last 28 years, and described the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T lymphocytes or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients in the United States. Most patients presented with enlarged lymph nodes and enlarged spleens;fever, hepatitis, and low blood cell counts were common. Most patients died of infection or EBV positive lymphoma. Unlike cases reported from Japan, we found that patients in the United States often had a progressive loss of B lymphocytes and low levels of immunoglobulins in the blood. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many patients in the United States had reduced NK cell numbers. While immunosuppressive medications, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active EBV positive lymphoma disease that was unresponsive to chemotherapy. The EBV genome encodes about 85 different proteins and the immune system produces antibodies to many of these proteins. We developed an assay, luciferase immuneprecipitation system (LIPS), to measure antibody response to 18 different EBV proteins. We found elevated levels of antibodies to 9 of these viral proteins in persons infected with the virus. The highest antibody levels were found to EBV proteins on the surface of the virus (gp350, gp42) and an EBV protein on the virus shell (p23 capsid protein). Antibodies were also detected to the viral DNA polymerase, an EBV transcription factor, another virus capsid protein, a protein expressed during latent infection (EBNA1), and a viral protein that blocks cell death. This study indicates that antibodies are produced to multiple EBV proteins.